Method of filtering platelets to remove antiplatelet and anticoagulant agents

ABSTRACT

The present invention relates to methods for removing antiplatelet agents and anticoagulants from a platelet preparation. In one embodiment, the method includes the step of flowing the platelet preparation through a filtering tube comprising a filtering membrane and separating the antiplatelet agents and anticoagulants from the platelet preparation by tangential flow filtration. In another embodiment, the method includes the step of passing the platelet preparation through porous material that specifically binds to the antiplatelet agents and anticoagulants.

This application claims priority from U.S. Provisional Application Ser.No. 61/187,052, filed Jun. 15, 2009 and claims priority from U.S.Provisional Application Ser. No. 61/282,306, filed Jan. 19, 2010. Theentirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

The present invention relates generally to preservation of plateletsand, in particular, to methods and devices for removing antiplateletand/or anticoagulation agents from stored platelets.

BACKGROUND

When blood vessels are damaged, cell fragments released from the bonemarrow, called platelets, adhere to the walls of blood vessels and formclots to prevent blood loss. It is important to have adequate numbers ofnormally functioning platelets to maintain effective clotting, orcoagulation, of the blood. Occasionally, when the body undergoes trauma,or when the platelets are unable to function properly, it is necessaryto replace or transfer platelet components of blood into a patient. Mostcommonly, platelets are obtained from volunteer donors either as acomponent of a whole blood unit, or via plateletpheresis (withdrawingonly platelets from a donor and re-infusing the remaining of the bloodback into the donor). The platelets then are transferred to a patient asneeded, a process referred to as “platelet transfusion”.

Platelet transfusion is indicated under several different scenarios. Forexample, an acute blood loss, either during an operation or as a resultof trauma, can cause the loss of a large amount of platelets in a shortperiod of time. Platelet transfusion is necessary to restore a normalability to control blood flow, or haemostasis. In a medical setting, anindividual can develop a condition of decreased number of platelets, acondition known as thrombocytopenia. The condition can occur as a resultof chemotherapy, and requires platelet transfusion to restore normalblood clotting.

Unlike red blood cells, which can be stored for forty-five (45) days,platelets can be stored for only five to seven days. The short storageterm, or shelf-life, of the platelets severely limits the useful spanfor a platelet supply. A consequence of this short shelf-life is thatplatelets must be collected close to their time of use, which makes itextremely difficult to coordinate platelet collection and plateletsupply.

One reason that platelets have such a short shelf-life is because theybecome activated during the process of collection. The activationprocess leads to externalization of platelet canalicular surfacesexposing receptor sites, such as GPIIb/IIIa. Phosphatidylserine residueson activated platelets tend to cause platelet aggregation, which resultsin cell death (i.e., apoptosis) upon re-infusion into patients. Thus, aplatelet functional half-life is significantly reduced.

Another reason that platelets have a short shelf-life is because aninadequate oxygen supply alters the metabolic activity of the platelets.In an environment lacking a sufficient oxygen supply, the plateletsundergo an anaerobic mechanism leading to accumulation of lactic acid.The increased concentration of lactic acid causes a drop in pH, andresults in cell death. Although platelets can be stored in gas permeablebags using a shaker bath under a stream of air to help overcome thisproblem, such storage methods are costly and extremely inefficient andinadequate in meeting the oxygen requirements of the stored platelets.

Platelet sterility is difficult to maintain because platelets cannot bestored at low temperatures, for example −80° C. to 40° C. As previouslymentioned, a low storage temperature for the platelets initiates anactivation process within the platelets that leads to aggregation andcell death. Nonetheless, bacterial growth in the platelet medium atsuitable storage temperatures, e.g., room temperature, can lead to anunacceptable occurrence of bacterial contamination in platelets used fortransfusion. As a result, the Food and Drug Administration (FDA) limitsthe storage time of platelets to five days, thereby safeguarding thetransfusion supply from bacterial contamination.

Antiplatelet agents and/or anticoagulants have been utilized for thepreservation of platelets. These preservative agents, when added tofreshly collected platelets, either by the huffy coat method or byaphaeresis, permit extended storage of platelets in a temperature rangeof −80° C. to 40° C., while maintaining the stability of platelets aswell as their functionality when transfused.

The antiplatelet agents and anticoagulants, however, need to be removedfrom the platelets storage bags prior to transfusion into the patient,thereby eliminating any concerns of adverse effects of the preservativeagents.

SUMMARY

One aspect of the present invention relates to a method for removingantiplatelet agents and anticoagulants from a platelet preparation. Themethod includes flowing the platelet preparation through a filteringtube comprising a filtering membrane and separating the antiplateletagents and anticoagulants from the platelet preparation by tangentialflow filtration.

In an embodiment, the filter membrane comprises a material selected fromthe group consisting of regenerated cellulose, cellulose acetate,polyamide, polysulfone, polyethylsulfone and combinations thereof.

In a related embodiment, the filter membrane comprises polysulfone orpolyethylsulfone.

In another embodiment, the filter membrane has a pore size ranging frommolecular cut off of 3000 daltons to 0.5 micron.

In another embodiment, the filtering tube has an inner diameter of atleast 0.5 mm.

Also disclosed is a method for removing antiplatelet agents andanticoagulants from a platelet preparation. The method includes passingthe platelet preparation through porous material that specifically bindsto the antiplatelet agents and anticoagulants.

In one embodiment, the porous material comprises a nanofiber.

In a related embodiment, the nanofiber is a cellulose nanofiber.

In another related embodiment, the cellulose nanofiber has a diameterbetween 5-60 nm.

In another related embodiment, the cellulose nanofiber is modified toprovide specific binding sites for a given antiplatelet agent or ananticoagulant.

In another embodiment, the porous material comprises a reinforcedcomposite film comprising 90% polyvinyl alcohol and 10% nanofiber.

In another embodiment, the nanofiber is a biodegradable nanofiber.

In a related embodiment, the biodegradable nanofiber comprisespoly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA),poly(lactic-co-glycolic acid) (PLGA), or combinations thereof.

In another related embodiment, the surface of the biodegradablenanofiber is chemically modified by oxygen plasma treatment and in situgrafting of hydrophilic acrylic acid (AA).

In another related embodiment, the biodegradable nanofiber forms aporous filter with a thickness of 200-800 nm, a pore size of 2-30micron, and a porosity of 94-96%.

In another embodiment, the nanofiber is a carbon nanofiber.

In a related embodiment, the carbon nanofiber is a chemically modifiedcarbon nanofiber.

Also disclosed is a method for antiplatelet agents and anticoagulantsfrom a platelet preparation using diafiltration. The method includescirculating the platelet preparation through a hollow fiber membranecapable of separating the antiplatelet agents and anticoagulants fromthe platelet preparation, wherein a diafiltration buffer is added to theplatelet preparation during circulation to maintain a constant volume ofthe platelet preparation.

In one embodiment, the hollow fiber membrane comprises a materialselected from the group consisting of regenerated cellulose, celluloseacetate, polyamide, polyurethane, polypropylene, polysulfone,polyethersulfone, polycarbonate, nylon, polyimide and combinationsthereof.

In another embodiment, the hollow fiber membrane comprises polysulfoneor polyethylsulfone.

In another embodiment, the hollow filter membrane has an inner diameterof at least 0.5 mm and a pore size ranging from molecular cut off of3000 daltons to 0.5 micron.

Also disclosed is a method for removing antiplatelet agents andanticoagulants from a platelet preparation. The method includes flowingthe platelet preparation through the surface of a filtering membrane andseparating the antiplatelet agents and anticoagulants from the plateletpreparation by tangential flow filtration.

In one embodiment, the filtering membrane has a pore size ranging frommolecular cut off of 3000 daltons to 0.5 micron.

Also disclosed is a filter for removing antiplatelet agents andanticoagulants from a platelet preparation. The filter comprises ananofiber modified to bind specifically to an antiplatelet agent or ananticoagulant.

In one embodiment, the nanofiber is selected from the group consistingof cellulose nanofibers, biodegradable nanofibers and carbon nanofibers.

BRIEF DESCRIPTION OF FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a diagram showing a typical continuous diafiltration system.

FIG. 2 is a thromboelastography (TEG) of platelets with inhibitorsbefore (black) and after plasma filtration (green).

FIG. 3 is a TEG of platelets with saline before (black) and after plasmafiltration (green).

FIG. 4 is a diagram showing the platelet response to TRAP in thepresence of the inhibitors.

FIG. 5 is a diagram showing the platelet response to TRAP after theremoval of the inhibitors.

FIG. 6 is a diagram showing the platelet response to collagen in thepresence of the inhibitors.

FIG. 7 is a diagram showing the platelet response to collagen after thediafiltration

FIG. 8 is a diagram showing an experimental platelet filtration system.

FIG. 9 is a thromboelastogram showing (black line) platelets withoutinhibitors and platelets after the antiplatelet agents had been removedwith 15 volume exchange (green line)

FIG. 10 is a diagram showing the platelet response to TRAP in theabsence of the inhibitors.

FIG. 11 is a diagram showing the platelet response to TRAP after theremoval of inhibitor by 15 volume exchange with Intersol.

FIG. 12 is a diagram showing the platelet response to collagen in theabsence of the inhibitors.

FIG. 13 is a diagram showing the platelet response to collagen after theremoval of inhibitor by 15 volume exchange with Intersol.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. Various modifications to thepreferred embodiments will be readily apparent to one skilled in theart, and the general principles defined herein may be applied to otherembodiments and applications without departing from the scope of theinvention. The present invention is not intended to be limited to theembodiments shown, but is to be accorded the widest possible scopeconsistent with the principles and features disclosed herein.

One aspect of the present invention relates to a method for removingantiplatelet agents and anticoagulants from a platelet preparation. Themethod includes the step of flowing the platelet preparation through afiltering tube comprising a filtering membrane and separating theantiplatelet agents and anticoagulants from the platelet preparation bytangential flow filtration.

Filtration is a pressure driven separation process that uses separationprocess that uses membranes to separate components in a liquid solutionor suspension based on their size and charge differences Filtration canbe broken down into two different operational modes—normal flowfiltration (NFF) and tangential flow filtration (TFF). In NFF, fluid isconvected directly toward the membrane under an applied pressure.Particulates that are too large to pass through the pores of themembrane accumulate at the membrane surface or in the depth of thefiltration media, while smaller molecules pass through to the downstreamside. This type of process is also called dead-end filtration.

In TFF, the fluid is pumped tangentially along the surface of themembrane. An applied pressure serves to force a portion of the fluidthrough the membrane to the filtrate side. As in NFF, particulates andmacromolecules that are too large to pass through the membrane pores areretained on the upstream side. However, in this case the retainedcomponents do not build up at the surface of the membrane. Instead, theyare swept along by the tangential flow. This feature of TFF makes it anideal process for finer sized-based separations. TFF is also commonlycalled cross-flow filtration. However, the term “tangential” isdescriptive of the direction of fluid flow relative to the membrane.

In one embodiment, the antiplatelet agents and anticoagulants areseparated from the platelet preparation by diafiltration, wherein adiafiltration buffer is added to the platelet preparation duringcirculation to maintain a constant volume of the platelet preparation.

Diafiltration is a TFF method of “washing” or removing permeablemolecules (impurities, salts, solvents, small proteins, etc) from asolution. Because it is a significantly faster and scalable method,diafiltration frequently replaces membrane tube dialysis. The success ofdiafiltration is largely determined by the selection of an appropriatemembrane. The membrane pores must be large enough to allow the permeablespecies to pass through and small enough to retain the larger species. Arule of thumb in selecting your first membrane is to choose a membranewhose pore size is rated 2-5× smaller than anything you are trying toretain and 2-5× larger than anything you are trying to pass through themembrane. A large variety of pore sizes are available in theultrafiltration and microfiltration range for this purpose.

The filtering membranes used in TFF are typically made of regeneratedcellulose, cellulose acetate or polyamide (as the filter composite onpolysulfone support), polysulfone or polyethylsulfone. These membranescome with a wide range of pore sizes. The effective pore size definesthe process and the particular application. Microfiltration refers tomembranes with nominal pore size from 0.05 micron to 1.0 micron.Microfiltration membranes retain intact cells and cell debris and passcolloidal materials, viruses, proteins and salts. Ultrafiltrationmembranes have a nominal molecular weight limit (NMWL) between 1 and1000 kD. They typically retain proteins and pass peptides and salts.Nanofiltration and reverse osmosis membranes have a NMWL of less than 1kD. They will retain antibiotics and most salts and pass water and somesalts.

These membranes can be chemically modified to provide a greater positiveor negative charge depending on the specific application therebyselectively binding a solute of interest. Alternatively, the surfacechemistry of these membranes can be modified to specifically bindsolutes of interest such as the antiplatelet agents or direct thrombininhibitors.

In certain embodiments, the membranes are hollow fiber membranes. TFFusing a hollow fiber membrane is accomplished by pumping the processsolution from a process reservoir into the inner diameter of a tubularfiber. The pores in the walls of the fiber allow the permeable speciesto pass through, while the larger species is retained in the bulk flow.The bulk flow then continues through to the ‘retentate’ end of the fiberand returns to the process reservoir that it was pumped out ofDiafiltration occurs by adding the replacement buffer or washingsolution to the process reservoir either at a rate equal to the permeateflow (continuous diafiltration) or by re-dilution after a certain levelof concentration (discontinuous diafiltration). Both methods result in adecrease in concentration of the permeable species while the retainedspecies remains in the solution that is gently circulating through thetangential flow system.

FIG. 1 shows a typical continuous diafiltration system in which thebuffer is automatically added to the process reservoir by vacuumsuction. It includes a pump (a), pressure measurement device (b), flowmeasurement device (c), process reservoir (d), buffer reservoir (e) andhollow fiber filter module (f). The pump circulates the process solutionfrom the process reservoir, through the filter and back to the processvessel at a controlled flow and shear rate. Pressure measurements areacquired in this re-circulation loop to control and record the drivingforce through the membrane. Careful measurement of the permeate flowrate enables accurate process scale up and process optimization.Diafiltration occurs simply by adding the diafiltration buffer to thiscirculation loop. Working with a hollow fiber module, tubing and anair-tight sealable bottle is a simple means of performing a continuousdiafiltration.

To begin the diafiltration in an airtight system, a vacuum needs to becreated in the process vessel. This can be accomplished by submergingthe buffer addition tube into a bottle of diafiltration buffer as shownin FIG. 1. As permeate flows out of the system, the vacuum in the sealedprocess reservoir pulls buffer into it at a flow rate equal to theprocess flux. When the target volume of diafiltration buffer has beencollected in the permeate vessel, the process is stopped simply bystopping the permeate flow and breaking the vacuum seal on the feedreservoir.

When airtight systems are not possible, particularly for pilot andmanufacturing scale processes, buffer addition can be controlled tomatch the permeate flow rate through the use of a single- ordouble-headed secondary pump adding buffer into the feed or processreservoir. Sometimes, it is advantageous to reduce the process volume byconcentration before diafiltration. There is a relationship between thevolume of buffer required to remove a permeable species and the productsolution volume in the process reservoir. By understanding thisrelationship, the cost associated with the process time and the volumeof buffer can be minimized.

Antiplatelet agents, as used hereinafter, refer to any agent thatreversibly impedes platelet activation and/or aggregation. Agents thatcan impede platelet activation and/or aggregation include, but are notlimited to, heparin, heparin substitutes, prothrombopenicanticoagulants, platelet phosphodiesterase inhibitors, dextrans, and thelike, or mixtures thereof. Examples of heparin and heparin substitutesinclude, but are not limited to, heparin calcium, such as calciparin;heparin low-molecular weight, such as enoxaparin and lovenox; heparinsodium, such as heparin, lipo-hepin, liquaemin sodium, and panheprin;and heparin sodium dihydroergotamine mesylate. Suitable prothrombopenicanticoagulants are, for example, anisindione, dicumarol, warfarinsodium, and the like. More specific examples of phosphodiesteraseinhibitors suitable for use in the invention include, but are notlimited to, anagrelide, dipyridamole, pentoxifyllin, and theophylline.Examples of dextrans are, for example, dextran 70, such as HYSKON®(CooperSurgical, Inc., Shelton, Conn., U.S.A.) and MACRODEX®(Pharmalink, Inc., Upplands Väsby, Sweden), and dextran 75, such asGENTRAN® 75 (Baxter Healthcare Corporation, Deerfield, Ill., U.S.A.).

Antiplatelet agents include, but are not limited to, active agents thatbind GPIIb/IIIa sites in a reversible manner and non-steroidalanti-inflammatory drugs (NSAIDs). In a preferred composition, the activeagents for binding to or associating with GPIIb/IIIa sites have acirculating half-life of inhibition of 4 hours or less. Examples ofsuitable antiplatelet agents for binding GPIIb/IIIa sites in areversible manner are eptifibatide (INTEGRILIN®, Schering-PloughCorporation, Kenilworth, N.J., U.S.A.), orbofiban, xemilofiban,Lamifiban, tirofiban, abciximab, XJ757, DUP728, XR299, linear or novelcyclic RGD peptide analogs, cyclic peptides, peptidomimetics andnon-peptide analogs conjugated to Nitric Oxide donor and the like, andmixtures thereof.

Non-steroidal anti-inflammatory drugs (NSAIDS) are commonly available,and typically are used for treating inflammation. Generally, NSAIDS canhave a salicylate-like or non-salicylate structure. NSAIDS suitable forthe invention can be salicylate-like or non-salicylate NSAIDS that bindreversibly and inhibit platelet aggregation in vitro, but are clearedrapidly, i.e. quickly eliminated from the body, when infused (typically,in less than about 2 hours). NSAIDS suitable for the invention include,but are not limited to, for example, salicylate-like NSAIDS, such asacetaminophen, carprofen, choline salicylate, magnesium salicylate,salicylamide, sodium salicylate, sodium thiosulfate, and the like, andmixtures thereof. Examples of non-salicylate NSAIDS include, but are notlimited to, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium,flurbiprofen, hydroxychloroquin, ibuprofen, indomethacin, ketoprofen,ketorolac tromethamine, meclofenamate sodium, mefenamic acid,naburnetone, naproxen, naproxen sodium, oxyphenbutazone, phenylbutazone,piroxicam, sulfinpyrazone, sulindac, tolmetin sodium, dimethylsulfoxide, and the like, and mixtures thereof.

Antiplatelet agents also include any agent that inhibits chemicalpathways within the platelets leading to reduction in plateletactivation. Typically, agents that inhibit chemical pathways leading toreduced platelet activation are calcium sequestering agents, such ascalcium channel blockers, α-blockers, β-adrenergic blockers, and thelike, and mixtures thereof. More specific examples of calciumsequestering agents include, but are not limited to, anticoagulantcitrate dextrose solution, anticoagulant citrate dextrose solutionmodified, anticoagulant citrate phosphate dextrose solution,anticoagulant sodium citrate solution, anticoagulant citrate phosphatedextrose adenine solution, potassium oxalate, sodium citrate, sodiumoxalate, amlodipine, bepridil hydrochloride, diltiazem hydrochloride,felodipine, isradipine, nicardipine hydrochloride, nifedipine,nimodipine, verapamil hydrochloride, doxazosin mesylate,phenoxybenzamine hydrochloride, phentolamine mesylate, prazosinhydrochloride, terazosin hydrochloride, tolazoline hydrochloride,acebutolol hydrochloride, atenolol, betaxolol hydrochloride, bisoprololfumarate, carteolol hydrochloride, esmolol hydrochloride, indoraminehydrochloride, labetalol hydrochloride, levobunolol hydrochloride,metipranolol hydrochloride, metoprolol tartrate, nadolol, penbutololsulfate, pindolol, propranolol hydrochloride, terazosin hydrochloride,timolol maleate, guanadrel sulfate, guanethidine monosulfate,metyrosine, reserpine, and the like, and mixtures thereof.

The anticoagulants include Xa inhibitors, such as DX-9065a, RPR-120844,BX-807834 and SEL series Xa inhibitor; IIa inhibitors such as DUP714,hirulog, Argobatran, and hirudin, and mixtures thereof; and otherpeptidomimetic or non-peptide Xa inhibitors, IIa inhibitors, or mixturesthereof. Some of these inhibitors are discussed in more details below.

In a preferred embodiment, the removal of antiplatelet agent and anticoagulants would involve the use of tangential flow filtration usingmicrofiltration membranes. Micro filtration membrane materials include,but are not limited to, regenerated cellulose, cellulose acetate,polyamide, polyurethane, polypropylene, polysulfone, polyethyl sulfone,polycarbonate, nylon, polyimide and combinations thereof. In oneembodiment, the microfiltration membrane is a hollow fiber membrane madeof polysulfone or polyethyl sulfone. In another embodiment, the filtermembrane tubes has inner diameter of 0.5 mm or greater with the membranepore size of 0.05 micron or larger. In another embodiment, the membranehas a pore size ranging from a molecular cut off of 3000 daltons to 0.5micron.

In another embodiment, the platelet preparation is passed through thehollow fiber membrane filter at flow rates ranging from 150 ml/minute to370 ml/minute. Theses flow rates provide acceptable shear forces from2000-s to 4000-s. An acceptable pump provides a wide range of flow ratesand also provides continuous monitoring of inlet, retentate, permeateand transmembrane pressures. In one embodiment, the pump is the KrosFlow II pump (Spectrum Labs, Rancho Dominguez, Calif.). A replacementfluid suitable for the removal of antiplatelet and anticoagulant agentswould be fluids that are used for the storage of platelets. Typically a10 to 15 volume exchange will result in the removal of better than 99%of the added agents. Typically, 45 to 100 μg of antiplatelet agent, suchas Eftifibatide, and 2.5 to 10 mg of anticoagulant, such as Argobatran,may be removed. Typically, a unit of platelets obtained by the buffycoat method would contain 3×10¹¹ platelets in approximately 300milliliters plasma or other suitable preservative solution. Plateletscollected by aphaeresis usually contain 5×10⁹ platelets in 250milliliters of plasma or other suitable fluid.

In another embodiment, the platelet preparation is passed through thehollow fiber filter in a diafiltration device at flow rates ranging from20 to 400 ml/min, preferably 150 to 400 ml/min. The hollow fibermembrane filters with a pore size ranging from molecular cut off of 3000daltons to 0.5 micron are acceptable. The preferred pore size is 0.05micron. For the exchange of one unit of platelets (300 to 400 ml) thepreferred surface area of the filtration module is 2500 cm². This alongwith a flow rate of 370 ml/min allows the complete removal (>99%) of theantiplatelet and anticoagulant agents contained in a unit of plateletsin 15 minutes. The diafiltration buffer can be any solution suitable forplatelet storage. In one embodiment, the diafiltration buffer is acommercially available platelet storage solution (T-Sol) with 20%plasma.

Another aspect of the present invention relates to a method for removingantiplatelet agents and anticoagulants from a platelet preparation. Themethod includes the step of passing the platelet preparation throughporous material that specifically binds to the antiplatelet agents andanticoagulants.

In certain embodiments, the porous material comprises a nanofiber.Examples of nanofiber include, but are not limited to, cellulosenanofibers, biodegradable nanofibers and carbon nanofibers.

Cellulose nanofibers may be obtained from various sources such as flaxbast fibers, hemp fibers, kraft pulp, and rutabaga, by chemicaltreatments followed by innovative mechanical techniques. The nanofibersthus obtained have diameters between 5 and 60 nm. The ultrastructure ofcellulose nanofibers is investigated by atomic force microscopy andtransmission electron microscopy. The cellulose nanofibers are alsocharacterized in terms of crystallinity. In one embodiment, the membranefilter is a reinforced composite film comprising 90% polyvinyl alcoholand 10% nanofibers.

The chemistry of these cellulose fibers can be modified to providespecific binding sites for a given antiplatelet agent and ananticoagulant. These fibers can be coated onto the surface of currentlyavailable disposable filter platforms like those used for sterilizingsmall volumes of fluids.

Biodegradable polymers, such as poly(glycolic acid) (PGA), poly(L-lacticacid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA), can be dissolvedindividually in the proper solvents and then subjected toelectrospinning process to make nanofibrous scaffolds. Their surfacescan then be chemically modified using oxygen plasma treatment and insitu grafting of hydrophilic acrylic acid (AA). In one embodiment, thebiodegradable nanofibrous scaffold has a fiber thickness in the range of200-800 nm, a pore size in the range of 2-30 micron, and porosity in therange of 94-96%.

The ultimate tensile strength of PGA will be about 2.5 MPa on averageand that of PLGA and PLLA will be less than 2 MPa. Theelongation-at-break will be 100-130% for the three nanofibrousscaffolds. When the surface properties of AA-grafted scaffolds areexamined, higher ratios of oxygen to carbon, lower contact angles andthe presence of carboxylic (—COOH) groups are identified. With the useof plasma treatment and AA grafting, the hydrophilic functional groupscan be successfully adapted on the surface of electrospun nanofibrousscaffolds. These surface-modified scaffolds provide the necessary sitesfor adding ligands specific to the binding of a given antiplatelet agentand anticoagulant.

There are several approaches that can be utilized to convert activatedcarbon into bioreactive fibers. An example is provided to demonstratethe ability of these modified carbon nanofibers to provide carboxylic,hydroxyl and other chemically reactive sites for the binding of anyligand of interest.

Carbon nanofibers (CNF) can be synthesized by chemical vapor deposition(CVD). Amino acids, such as alanine, aspartic acid, glutamic acid andenzymes such as glucose oxidase (GOx) can be adsorbed on CNF. Theproperties of CNF (hydrophilic or hydrophobic) are characterized by thepH value, the concentration of acidic/basic sites and by naphthaleneadsorption. These fibers are readily amenable to crosslinking withligands f interest, i.e., the ability to selectively bind toantiplatelet agents and anticoagulants.

Another aspect of the present invention relates to a filtering tube forremoving antiplatelet agents and anticoagulants from a plateletpreparation. The filtering tube has an inner diameter of at least 0.5 mmand comprises a filter membrane with a pore size ranging from molecularcut off of 3000 daltons to 0.5 micron.

Another aspect of the present invention relates to a filter for removingantiplatelet agents and anticoagulants from a platelet preparation. Thefilter comprises a nanofiber that is modified to bind specifically to anantiplatelet agent or an anticoagulant.

In a related embodiment, the nanofiber is one of a cellulose nanofiber,a biodegradable nanofiber, and a carbon nanofiber.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

Example 1 Removal of Integrilin Using Diafiltration

A standard UV absorbance curve at 214 nm with Integrilin concentrationsranging from 3 to 50 μg/ml was established based on the followingmeasurements:

Integrilin Concentration O.D 214   50 ug/ml 0.710   25 ug/ml 0.532 12.5ug/ml 0.299 6.25 ug/ml 0.126 3.125 ug/ml  0.015

A test fluid containing 50 μg/ml Integrilin was circulated through ahollow fiber filter (Spectrum Laboratories X20S-300-O2S) at acirculation rate of 100 ml/min. Briefly, the test fluid was placed in anair tight container with a starting OD214 of 0.710. As the test fluidcirculated through the hollow fiber filter, the retentate volumedecreased. The lost fluid volume was replaced with fresh fluid from asecond container. After about a 10 minute circulation and a six volumeexchange, the OD214 of the test fluid was less than 0.01.

Example 2 Removal of Inhibitors from Platelet Concentrate

In one experiment, platelet concentrates obtained by the buffy coatmethod were used for the study. 48 micrograms of Integrilin(Eptifibatide), a GPIIb/IIIa inhibitor and 2.4 mg of Argabotran, asynthetic thrombin inhibitor, were added to 350 ml of platelets. Removalof the inhibitors was initiated by diafiltration. 15 ml of the plateletconcentrate was diafiltered against 60 ml of a solution containing 20%fresh frozen plasma in a commercially used platelet storage solution(T-Sol). The samples were stored overnight and platelet functionalitywas measured on Day 2, by Thromboelastography (TEG) and using standardagonists such as TRAP and Collagen. FIG. 2 shows the TEG scan of thetest sample before (black line) and after diafiltration (green line).FIG. 3 shows the TEG scan of a control sample (platelets with saline)before (black line) and after diafiltration (green line). The result inFIG. 3 suggests that most inhibitors had been removed by diafiltration.FIG. 4 shows the response to TRAP in the presence of the inhibitors.FIG. 5 shows the response to TRAP after the removal of the inhibitors.FIG. 6 shows the response to collagen in the presence of the inhibitorsand finally FIG. 7 shows the response to Collagen after thediafiltration.

In another experiment, Integrilin and Argabotran were added to plateletunits at three times the therapeutic concentrations (i.e., 48 microgramsfor Integrilin and 2.4 mg for Argatroban in 350 ml of platelets). Priorto the addition of the inhibitors, baseline data of plateletfunctionality was obtained. These baseline data includedthromboelastography, which assesses overall platelet function and clotstrength, as well as the TRAP test and the collagen test, which areadditional markers of platelet functionality.

The diafiltration was conducted in 40 ml aliquots using an airtight 50ml conical flask, a polysulfone hollow fiber cross flow module with asurface area of 240 cm² (FIG. 8). A sequential exchange with increasingvolumes of standard platelet additive solution (Intersol), indicatedthat a 15 volume exchange provided optimum results with the inhibitors.The pore size of the hollow fiber membrane selected was 0.05 micron. Thepore size can range from 3000 daltons molecular weight cut off to 0.5micron. The 15 volume exchange can be conducted with any currentlyavailable additive solution used for storing platelets.

Following the 15 volume exchange, homologous fresh frozen plasma wasadded to the platelets to achieve a plasma concentration of 25% (v/v).This is required because the plasma provides the soluble components ofcoagulation, thereby permitting the functionality tests performed.

The recirculation rate of the platelets (in Intersol containing 30%plasma) was set at 370 ml/minute. This was calculated to create a shearforce of approximately 4000-s. This shear force has been shown not toactivate platelets.

The Inlet pressure over three experiments was 8.13 psi, the retentatepressure was 6.15 psi. The pressure differential was 2 psi. The permeatepressure was essentially 0 and the transmembrane pressure was 7.15 psi.These pressures remained very constant throughout the exchange,indicating no fouling of the membrane. The permeate flow rate was around26 ml/minute.

When Argatroban is present, it completely blocks the activation ofplatelets and hence on the thromboelastogram, one sees a straight line.FIG. 9 shows the thromboelastograms of platelets without the inhibitors.The R value, i.e., the time it takes before the clot starts to form, asindicated by the splitting of the line, averaged 10.7 minutes. Followingthe 15 volume exchange, the average R value was 10.75. In a total of 4experiments conducted, the R value after 15 volume exchange was equal toor less than the baseline R value. Based on the thromboelastogram, atleast a 99.99% removal of inhibitors was achieved.

The maximum amplitude, MA, was unchanged from baseline, when the slightdilution of the platelets during the 15 volume exchange is taken intoconsideration. This parameter reflects the removal of Integrilin.

FIG. 10 shows the results of the Trap assay run on the platelets priorto the addition of the inhibitors. The area under the curve is expressedas standardized units. The control had a value of 67. The same assay runon the platelets after 15 volume exchange with Intersol was 68 (FIG.11). This assay is more sensitive to the presence of Integrilin. Ittherefore indicates essentially complete removal of this inhibitor.

The functionally of the platelets was also tested using collagen as theactivator. The baseline value for the platelets was 6 (FIG. 12). Thevalue obtained for the platelets following the removal of the inhibitorswith 15 volume exchange with Intersol was also 6 (FIG. 13).

The experiment described above is a method by which complete replacementof plasma in platelet concentrates, whether collected by apheresis,buffy coat or any other method, can be readily accomplished.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following embodiments. Theembodiments are intended to cover the claimed components and steps inany sequence which is effective to meet the objectives there intended,unless the context specifically indicates the contrary.

What is claimed is:
 1. A method for removing antiplatelet agents andanticoagulants from a platelet preparation, comprising: flowing aplatelet preparation comprising an antiplatelet agent, an anticoagulantand platelets through a filtering tube comprising a filtering membraneand separating the antiplatelet agent and anticoagulant from theplatelet preparation by tangential flow filtration, wherein theantiplatelet agent comprises a GPIIb/IIIa inhibitor and has acirculating half-life of inhibition of 4 hours or less and wherein thefilter membrane has a pore size ranging from molecular cut off of 3000daltons to 0.5 micron and wherein the anticoagulant comprises ashort-to-ultrashort acting factor Xa inhibitor.
 2. The method of claim1, wherein the filtering tube has an inner diameter of at least 0.5 mm.3. The method of claim 1, wherein the platelet preparation flows throughthe filtering tube at a flow rate of 150 to 400 ml/min.
 4. The method ofclaim 1, wherein an extraction liquid is circulated outside thefiltering tube in a counter current manner.
 5. The method of claim 4,wherein the extraction fluid comprises 0.9% w/v sodium chloride.
 6. Themethod of claim 1, wherein the filtering membrane is made of a materialselected from the group consisting of regenerated cellulose, celluloseacetate, polyamide, polyurethane, polypropylene, polysulfone,polyethersulfone, polycarbonate, nylon, polyimide and combinationsthereof.
 7. The method of claim 1, wherein the filtering membrane ismade of polysulfone or polyethylsulfone.
 8. A method for removingantiplatelet agents and anticoagulants from a platelet preparation,comprising: flowing a platelet preparation comprising an antiplateletagent, an anticoagulant and platelets along the surface of a filteringmembrane and separating the antiplatelet agent and anticoagulant fromthe platelet preparation by tangential flow filtration, wherein theantiplatelet agent comprises a GPIIb/IIIa inhibitor and has acirculating half-life of inhibition of 4 hours or less and wherein thefilter membrane has a pore size ranging from molecular cut off of 3000daltons to 0.5 micron and wherein the anticoagulant comprises ashort-to-ultrashort acting factor Xa inhibitor.
 9. The method of claim8, wherein the filtering membrane is made of a material selected fromthe group consisting of regenerated cellulose, cellulose acetate,polyamide, polyurethane, polypropylene, polysulfone, polyethersulfone,polycarbonate, nylon, polyimide and combinations thereof.
 10. The methodof claim 8, wherein the filtering membrane is made of polysulfone orpolyethylsulfone.